Isabel Pizarro a, Milagros Gómez-Gómez b, Jennifer León a, Domingo Román a, M.

Antonia Palacios b,⁎

a
Facultad de Ciencias Básicas, Universidad de Antofagasta, 02800 Antofagasta, Chile
b
Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, 28040 Madrid, Spain

HIGHLIGHTS

• Vegetables from contaminated soils of Chiu Chiu (Chile) present high As levels. • Only
toxic inorganic species of As (III and V) are present in carrots and quinoa.
• No transformations of As species during in vitro gastrointestinal digestion.
• Estimated dietary intake and exposure of As have been calculated.

article info abstract

Article history: Consumption of vegetables grown in arsenic (As)-contaminated soils is an important exposure route to the element for humans.
Received 17 February 2016 The present study is focused on locally-grown, frequently-consumed vegetables, such as carrots (Daucus carota), beets (Beta
Received in revised form 29 April 2016 vulgaris) and quinoa (Chenopodium) from the As-polluted Chiu Chiu area in Northern Chile. The latter region is affected both
Accepted 29 April 2016 Available by As discharge from copper mining activity and natural As contamination, leading to a high As content in local food and water.
online xxxx For the selected vegetables, the following aspects were investigated: i) Their total As, Cu, Pb, Cr, Cd and Mn content; ii) Arsenic
speciation in the edible part of the vegetables by liquid chromatography inductively-coupled plasma mass spectrometry (LC-
Editor: D. Barcelo ICPMS) analysis; iii)

Keywords: Arsenic bioaccessibility in the vegetables during in vitro gastrointestinal digestion; iv) Arsenic species present in the extracts
As obtained from in vitro gastrointestinal digestion; and v) Arsenic dietary exposure estimates for the assessment of the risk posed
Gastrointestinal digestion by the vegetables consumption.
Vegetables
A significant degree of As contamination was found in the vegetables under study, their metal content having been compared
Estimated dietary intake
with that of similar Spanish uncontaminated products. In vitro gastrointestinal digestion of the studied vegetables led to
quantitative extraction of As from carrots and beets, whereas efficiency was about 40% for quinoa. For carrots, only As(III)
and As(V) species were found, being their concentration levels similar. In the case of quinoa, around 85% of the element was
present as As(V). For beets, inorganic As(V) and unknown overlapped As species (probably arsenosugars) were found. No
significant transformation of the original As species was observed during in vitro gastrointestinal digestion. Arsenic dietary
exposure values obtained for the three vegetables (0.017–0.021 μg As person−1 day−1) were much lower than the JFCFA's
safety limit of 50 μg As person−1 day−1. Therefore, no toxicological risk would be expected from the intake of these
vegetables.
© 2016 Published by Elsevier B.V.

1. Introduction
Since 1915, the Northern Chilean economy has been mainly supported by
the exploitation of copper mining resources and mineral smelting plants.
Arsenic can be present in minerals, associated with copper and other heavy
metals, e.g. being present in enargite (Cu3AsS4). During the pyrometallurgical
process, As is released as As2O3 both in the form of gas phase and as fine
particles (Soriano et al., 2012). In
⁎ Corresponding author.
E-mail address: palacor@ucm.es (M.A. Palacios).
http://dx.doi.org/10.1016/j.scitotenv.2016.04.199 0048-
9697/© 2016 Published by Elsevier B.V.
addition, the Loa river water is also naturally contaminated by As. The
concentration of As in the water of the Loa hydrographic basin changes
dramatically over the course of the river. This can vary from no contamination
558 I. Pizarro et al. / Science of the Total Environment 565 (2016) 557–563
in the Copiapó river (snow melting, concentration b0.005 mg L−1) to extreme
contamination in the San Salvador river (2.0–2.5 mg L−1) (Sträter et al., 2010;
Pizarro et al., 2010). Between 1958 and 1970, the Antofagasta population was
drinking water from the Loa river with an As concentration of about 1000 μg L−1
(most probably in the form of As(V)). Successive improvements in the water
treatment resulted in an annual average value of 50 μg As L−1, unfortunately with
some areas still presenting levels well above the average. These As levels are
still far from the World Health Organization (WHO) maximum recommended
levels of 10 μg L−1 (WHO, 1996). Therefore, natural geological As contamination
of water, anthropological action, the area desert climate, and the exposure of
organisms to this high As contamination, result in very extreme conditions for
life. This is similar to other situations in well-known parts of the world
contaminated with As such as India, China or New Zealand (Ahsan and Del
Valls, 2011; Huang et al., 2014; Shakoor et al., 2015; Rahman et al., 2014a,
2014b). Contamination of As and other heavy metals has been reported in soil,
water and vegetables growing in the Chilean II Region (Pizarro et al., 2003b;
Flynn et al., 2002).
Cancer and other diseases associated with As exposure reach alarming levels
in this Chilean area. Recent studies have shown the presence of As(III) in the
auricle and saphena of the people living in the Chiu Chiu area, suggesting that
As could be involved in cardiovascular disease development (Roman et al.,
2011). Bronchial, lung, bladder diseases and renal cancer have an alarmingly
unusual high incidence in this region (Steinmaus et al., 2013; Ferreccio et al.,
2013). Assessment of the contribution to human diet of As and other contaminant
elements in food requires studying not only their total element content but also
their absorption rates in the gastrointestinal tract. A way to evaluate the
absorbable fraction in trace element bioaccessibility studies is the “in vitro”
simulation of the digestion process. In the case of As, it is also important to
discern its species present in food and their possible transformation during the
digestion process (Hur et al., 2011; Khouzam et al., 2011, Calatayud et al., 2013).
Inorganic arsenic species are the most dangerous forms of the element in
food, being As(III) more toxic than As(V). The formation of As(III) from As(V)
species present in natural environmental conditions, can be attributed in most of
the cases to a detoxification mechanism taking place in microorganisms but also
in more complex organisms, where reduction occurs prior to methylation. The
methylation process first generates monomethylarsonic acid (MMA) and then
dimethylarsinic acid (DMA), both being less toxic than inorganic species
(Cullen, 2014).
Vegetables, and in particular rice, growing in natural or contaminated soils
are one of the most interesting food samples, from the toxicological point of
view, for As bioaccessibility and speciation studies. Rice presents the highest
concentrations of inorganic arsenic species as compared to other tested products
(Tyson, 2013; Rahman et al., 2014a, 2014b; He et al., 2012). Broccoli, lettuce,
potatoes or carrots, can also accumulate As when the soil or the irrigation water
contains As(V) (Johnson et al., 2010).
The present study is focused on the determination of As (and its implications
for human health) in carrots, beets and quinoa from the polluted Chiu Chiu area
located in Northern Chile (about 10 km from Calama and 235 km from
Antofagasta city). The following issues were investigated: i) Determination of
total content of As and other metals from mining activities (Cr, Cu, Pb, Mn and
Cd) in the edible parts of carrots, beets and quinoa grown in the area where the
indigenous population live; ii) As species present in the edible part of these
vegetables; iii) Arsenic bioaccessibility in the vegetables under in vitro
gastrointestinal digestion; iv) Arsenic species present in the in vitro
gastrointestinal extracts; and vi) Evaluation of the health risk posed by the As
dietary exposure estimates.
2. Materials and methods
2.1. Instrumentation
An ICPMS, (HP-4500, Agilent Technologies, Analytical System, Tokyo,
Japan) operating under normal multi-element tuning conditions was used for
total arsenic and other metals analysis. The main analytical parameters of the
ICPMS were the following: R.F. forward power, 1350 W; reflected power, 2.2
W; plasma argon flow rate, 14 L min−1; auxiliary argon flow rate, 0.9 L min−1;
nebulizer argon flow rate 0.85 L min−1, integration time, 0.1 s per point; points
per peak, 3.
For arsenic speciation, an ICPMS has been used as detector system after
LC species separation. The column effluent was directly introduced into a
Meinhard-type concentric glass nebulizer and a double-pass Scott-type spray
chamber with a Peltier cooling system set at 5 °C. Data was collected by single
ion monitoring at m/z 75. For chromatographic separation, a high-pressure
pump (LDC Division, Riviera Beach, Florida, USA) was used as a sample
delivery system. Samples of 100 μL were introduced through a 0.45-μm nylon
syringe filter into an injection valve (Rheodyne 9125, USA).
Polytetrafluoroethylene tubing (i.d. 0.5 mm) connections were used for
coupling the LC system to ICP-MS. A Hamilton PRP-×100 LC column (10
μm, 250 mm × 4.1 mm) Torrance, CA, USA) with a Phenomenex precolumn
(25 × 2.3 mm 12– 20 μm) was used.
All signal quantification was performed in the peak area mode. Peaks were
integrated usingeither ICPMS Chemstation software (AgilentTechnologies)
or Grams/32 software (Galactic Industries, Salem, NY, USA).
A Sonopuls Ultrasonic Homogenizer (HD 2200. Bandelin Electronic,
Berlin, Germany), a Vibromatic rocking mixer (Selecta, Barcelona, Spain)
and a Gyrozen 416 Centrifuge (Controltecnia Instruments, Barcelona, Spain)
were also used in the sample extraction process.
2.2. Reagents
A standard solutions of As(III) of 1000 mg L−1 TraceCERT™ Ultra (Fluka,
Sigma-Aldrich, Steinheim, Germany) and As(V) 1000 mg L−1 CertiPUR
(Merck, Darmstadt, Germany), both prepared in HNO3 (2% v/ v), were used.
Dimethylarsinic acid (DMA) and methylarsonic acid (MMA) standard
solutions of 1000 mg L−1, were prepared from methyl disodium arsenate
(Na2CH3AsO3, 99% Supelco, Bellefonte, PA, USA) and cacodylic sodium
trihydro ((CH3)2AsNaO2·3H2O) 98% Fluka) TraceCERT ™. Arsenobetaine
(AsB) and arsenocholine (AsC) were obtained from Tri Chemical Laboratory
Inc. (Japan). Cd (Fluka), Cu (Fluka), Mn (Sigma Aldrich), Cr (Merck), and
Pb (Sigma Aldrich) standard solutions of 1000 mg L−1 were supplied in HNO3
(2% v/v). All solutions were kept at 4 °C in the dark. Working solutions were
daily prepared by dilution with Milli-Q water.
Analytical grade reagents were exclusively used. HNO3 60%, HCl 15%,
HClO4 70% and ammonium phosphate 98% were purchased from
Scharlab. Alpha-amylase (11,800 USP Units), pepsine (enzymatic activity
944 U/mg protein), pancreatin (activity equivalent to 4 × US Pharmacopoeia
specifications/mg pancreatin), lipase (944 USP units), protease (11,800 USP
units), bile extracts (glycine and taurine conjugates of hyodeoxycholic acid
and other bile salts) were all purchased from Sigma Aldrich (Spain).
Deionized water (18.2 MΩ cm), obtained with a Milli-Q system (Millipore,
Inc., Spain) was used for all solutions preparation. Plastic and glassware were
maintained in 10% HNO3 for 24 h before use.
2.2.1. Simulated gastrointestinal juices
Salivary juice (pH 6.8 ± 0.1) was prepared by dissolving 0.15 g NaH2PO4.
H2O, 0.029 g amylase, 0.1 g KCl and 0.1 g NaCl and made up to 100 mL with
water. Gastric juice (pH 1.8 ± 0.1) was prepared by dissolving 6.0 g of pepsin
and 0.88 g sodium chloride (Sigma) in water; pH was then adjusted with HCl
and the volume was made up to 100 mL with water. Intestinal juice was
prepared just before use by mixing equal volumes of A and B solutions.
(Solution A: 1.5 g pancreatin, 0.44 g sodium chloride, 30 mg alpha-amylase
and water up to 50 mL. Solution B: 2 g bile salts, 0.44 g sodium chloride and
water up to 50 mL) (Hur et al., 2011).
2.3. Carrots, beets and quinoa samples
Chilean vegetable samples were collected from 3 different places of the Chiu
Chiu Chilean area (Supplementary material file, Fig. S1).
 I. Pizarro et al. / Science of the Total Environment 565 (2016) 557–563
About 10 kg of carrots, beets and quinoa were taken. For comparison, about
10 kg of the same type of vegetables of Spanish origin were purchased. The
samples were sent fresh from origin to laboratory and kept frozen under
nitrogen atmosphere.
2.4. Certified reference materials (CRMs)
Rice flour (1568a NIST), white cabbage (BCR 679) and tomato leaves
(NIST 1573a) were used as certified reference materials. The concentration of
As and other heavy metals was determined on these CRMs following the same
method as the one described below for the vegetables under study.
2.5. Sample treatment for ICPMS determination of As, Cr, Cu, Mn, Pb and
Cd content in vegetables
Carrots and beets were peeled with a plastic knife and the edible part was
treated in a titanium blender until a homogeneous mash was achieved. A
fraction of about 2 kg of flesh were lyophilized, bottled and maintained at 4
°C until analysis. No other treatment apart from drying at 80 °C and storing
at 4 °C was performed on the quinoa.
For sample mineralization, about 0.5 g of the vegetables were digested in
PTFE vessels with 5 mL of concentrated HNO3 and 2 mL of H2O2. The
mixtures were kept for 4 h in open air until vapor production stopped,
followed by microwave-digestion under the following conditions: 600 W, 200
°C, 20 min and 150 psi (Enamorado–Báez et al., 2013). The digested samples
were transferred to polyethylene containers, diluted with 0.2 M HCl to
appropriate volumes and stored at 4 °C until analysis by ICPMS. Reagent
blanks showed no significant contamination.
Performance parameters of the analytical method were studied, being the
detection limits (DL) for the elements (expressed in μg kg−1) the following:
As: 5.0; Cd: 0.03; Cu: 0.1; Pb: 0.08; Mn: 2.7; Cr: 2.0. Relative standard
deviation (RSD) was within the 2–6% range depending on the element.
Method validation was performed in triplicate with the CRMs mentioned in
Section 2.4.
In order to check the possible interference of chloride in the 75As signal
due to the formation of 40Ar35Cl+ during ICPMS analyses, the signal at m/z 75
of salivary, gastric and intestinal juices was monitored. This signal intensity
for the three extracts was negligible and did not differ from that of a blank
solution, indicating that the procedure is free of interference by chloride.
2.6. Extraction procedures for arsenic speciation in the vegetables by
LCICPMS
For arsenic speciation by LC-ICPMS, two extraction procedures have
been employed.
2.6.1. Methanol:water (1:1) extraction
5 mL of methanol:water (1:1) solution was added to 0.5 g of either the
lyophilized carrots and beets or dried quinoa. The mixture was heated in a
thermostatic water bath for 30 min, at 55 °C, followed by sonication with an
ultrasonic probe for 5 min at 30% power and centrifugation at 2700g for 20
min at room temperature. Supernatants were collected and the extraction
procedure repeated in the solid fraction. The two liquid extracts were pooled
and evaporated to a final volume of 2 mL
(Pizarro et al., 2003a).
2.6.2. Enzymatic protease-lipase extraction
10 mg alpha-amylase and 3 mL water were added to 0.3 g of either the
lyophilized carrots and beets or fresh quinoa. The mixtures were sonicated
with an ultrasonic probe for 60 s at 30% power in an ice bath. After that, 30
mg protease was added and the mixture was again sonicated for 120 s and
centrifuged for 10 min at 2700g. The solution was made up to 2 mL for
speciation analysis (Guzman et al., 2009). The two different procedures were
performed in triplicate.
559
The chromatographic conditions for As species separation from both
procedures can be seen in Table S1 (Supplementary material file).
Arsenic species were identified in the different extracts by comparison of the
retention times from the respective LC-ICPMS chromatograms with those
observed by injection of standard solutions of the six arsenic species (As(III),
As(V), MMA, DMA, AsB and AsC). Furthermore, spiking experiments of the
extracts with each specific As species were also performed for confirmation of
the presence of these species. Arsenic species were quantified by measurement
of their chromatographic peak areas. No matrix effects were observed, with no
significant differences found between the results obtained by external calibration
and standard addition method.
2.7. In vitro gastrointestinal digestion of vegetables for arsenic bioaccessibility
evaluation
Samples were digested following a laboratory-simulated three-step digestion
process (Khouzam et al., 2011).
2.7.1. Salivary digestion
About 1.0 g of lyophilized sample was mixed with 5 mL of salivary juice and
2 mL water in a 25 mL erlenmeyer and shaked in a vibromatic JP Selecta (508
U/min) for 5 min for degassing. The mixture was heated for 5 min in a
thermostatic water bath (37–39 °C), shaked for 5 min, heated again for 5 min and
centrifuged at 3200g for 20 min to separate salivary and solid phases.
2.7.2. Gastric digestion
10 mL of the prepared gastric juice, was added to the solid fraction after
salivary digestion, heated for 1 h in a water bath, shaked for 30 min in a
vibromatic JP Selecta (508 U/min), adjusting the pH to 1.8 with HCl (15%), and
heated for additional 3 h at 36–39 °C. The mixture was centrifuged at 3200g for
20 min to separate the soluble and solid fractions.
2.7.3. Intestinal digestion
10 mL of the prepared intestinal juice was added to the solid fraction obtained
in the gastric digestion and the digestion procedure was repeated. The residue
was discarded.
The three liquid extracts after digestion were mineralized in a microwave
oven following the same procedure to that indicated in Section 2.5, and
appropriately diluted before analysis.
3. Results and discussion
3.1. As and Cr, Cu, Mn, Pb and Cd analysis in carrots, beets and quinoa
When vegetables are grown in contaminated soils, the bioavailable fraction
of the elements present in the soil may migrate to the vegetable through intake
mechanisms where microorganisms and the vegetal physiology are frequently
involved (Intawongse and Dean, 2008; Xiong et al., 2014; Nearing et al., 2014).
Table 1 shows the concentration of As, Cd, Cu, Pb, Mn and Cr in the
lyophilized flesh of carrots, beets and in dried quinoa growing in the Chiu Chiu
area; the concentration of the same elements in local vegetables from Spain; and
the metal concentration ratio between Chilean and Spanish samples. The
concentrations of all tested elements in the Spanish samples fall within those
levels reported for uncontaminated vegetables (Ramalli et al., 2005).
A preliminary study performed for As in the soils of three places where
vegetables were grown, showed an As content within the 48– 97 mg kg−1 range.
Interchangeable and oxidized As fractions were higher than 40% of the total
content found in the soils (data not shown). These fractions could be responsible
for the contamination of As in the three vegetables tested in comparison with
those of Spanish origin. Concentration level ratios for As between Chilean and
Spanish samples were of 26, 31 and 20 for carrots, beets and quinoa, respectively.
These high ratios have not been found for the rest of the analyzed elements.
The arsenic concentrations found in lyophilized carrots, beets and quinoa
were 0.52, 0.62 and 0.20 mg kg−1, respectively. For raw rice, the most studied
vegetable, the As concentration permitted by the Codex Alimentarious is 0.3 mg
560 I. Pizarro et al. / Science of the Total Environment 565 (2016) 557–563
kg−1 for total As and 0.2 mg kg−1 of inorganic As (Codex Alimentarious, 2013).
The WHO recommendation for rice is a limit of 1.0 mg As kg−1 (WHO, 1996).
Chile has established a maximum of 0.5 mg As kg−1 for cereals and legumes and
the same limits are permitted for rice (RSA, 2011). Therefore the concentration
found in the analyzed vegetables did not greatly exceed those levels allowed by
the Chilean law.
Previous studies performed a decade ago in carrots growing in the same area
showed total As levels around 50 mg kg−1, a hundred times higher than those
obtained nowadays (Pizarro et al., 2003b). Fortunately, soil cleaning and water
decontamination treatments carried out nowadays, could be responsible for the
decreasing trend found.
The methodology employed for the analysis of most of the elements has been
validated with CRMs of vegetables (Supplementary material file, Table S2).
3.2. Arsenic speciation in carrots, beets and quinoa
Although As(V) is the most common species in As-contaminated soils and
water, As(III) is in most cases the main arsenic species present in terrestrial
vegetables. Reduction of As(V) to As(III) is believed to be an efficient way for
the plants to protect themselves from the interruption of oxidative
phosphorylation exerted by As(V) (Finnegan and Chen, 2012). Organic arsenic
species can also be found in lower concentration levels (Ye et al., 2012).
In the extraction process of hydro-soluble species such as inorganic As,
MMA and DMA species, a water:methanol (1:1) mixture provides quantitative
recoveries and good species stability (Pizarro et al., 2003a). This extraction
method has commonly been used for inorganic As(III) and As(V), methylated
species and also arsenosugars in fresh seaweeds (McSheehy and Szpunar, 2000).
However, enzymatic hydrolysis is required for the extraction of species strongly
As, Cd, Pb, Cu, Mn and Cr content in Chilean and Spanish lyophilized vegetables. Results are expressed as mean ± SD (mg kg −1) for n = 3 determinations.
Vegetables As Cd
Carrots Chilean 0.52 ± 0.04 0,05 ± 0.01
Spanish 0.02 ± 0.01 0.07 ± 0.01
Ratio 26 0.7
Beets Chilean 0.62 ± 0.05 0.09 ± 0.01
Spanish 0.020 ± 0.003 0.05 ± 0.01
Ratio 31 1.8
Quinoa Chilean 0.20 ± 0.02 0.38 ± 0.07
Spanish 0.010 ± 0.002 bDL
Ratio 20 – 0.4
Ratio: metal concentration ratio between Chilean and Spanish samples.
Cr was below the DL in all samples analyzed.
bound to biomolecules (Guzman et al., 2009).
Table 2 shows the extraction efficiency achieved for total As in the vegetables
under study in the water:methanol (1:1) extract and in the enzymatic alpha-
amylase/protease extract. The contribution (%) of each As species detected by
LC-ICPMS to the total As content in both extracts is also displayed. In the case
of the analyzed carrots and quinoa, the use of an extraction mixture of
water:methanol (1:1) was appropriate, providing quantitative recoveries.
However, the possible presence of arsenosugar by-product species in lyophilized
beets, makes enzymatic hydrolysis necessary for their total extraction.
In carrots, only inorganic As(III) and As(V) species were found, at similar
concentration levels. For quinoa, 82% of the As was present as
Table 1
As(V), being about 15% as As(III). No other species were found in the
water:methanol (1:1) fraction for both vegetables. Terrestrial plants in general
are known to present very low efficiency mechanisms of biomethylation.
Probably the contamination of these soils makes it difficult for soil
microorganisms to develop biomethylation mechanisms that might produce
methylated species available for absorption through the vegetables roots (Ye
et al., 2012). Beets showed a different behavior, with inorganic As(V) (about
20%) and several unknown As species (representing about 70% of the total
As), with retention time of 4.5 min, being present (data not shown). Sucrose
is the sugar extracted from beets, therefore As-sucrose by-products might be
the unidentified species.
Previous studies performed in contaminated carrots grown in the same
area (Pizarro et al., 2003b) revealed that their As content mainly consisted of
toxic species, As(III) and As(V), accounting for 45% and 31% of the total As,
respectively. Some organic species of As were also present in very low
concentration. The results obtained in the present study, show a lack of
methylated species probably due to the lower arsenic concentration than that
previously found. However, the proportion of As(III) and As(V) species are
in a similar range.
3.3. Bioaccessibility of As in carrots, beets and quinoa and As speciation after
“in vitro” gastrointestinal digestion
Dynamic “in vitro” gastrointestinal models mimic the gradual transit of
ingested compounds through the digestive tract. Compared to in vivo studies,
the in vitro sequential gastrointestinal fluid extraction is a relatively simple,
yet effective approach to estimate the bioaccessibility of dietary arsenic (He
et al., 2012). Knowledge on the oral As bioaccessibility and the As species
present in food which could be released to the bloodstream is key for
estimating potential human health risks. Table 3 shows: the total As
concentration in the different extracts of the in vitro gastrointestinal digestion
process; the contribution of As species to the total As content in each
gastrointestinal extract; the total As extracted; and the As bioaccessibility.
Pb Cu Mn
0.12 ± 0.02 7.8 ± 0.9 4.8 ± 0.5
0.09 ± 0.01 8.8 ± 0.6 2.3 ± 0.2
1.3 0.9 2
0.36 ± 0.01 6.7 ± 0.9 8.4 ± 0.2
bDL(0.006) 3.8 ± 0.6 3.8 ± 0.2
– 1.7 2.2
0.04 ± 0.02 7.9 ± 0.7 14 ± 2
0.09 ± 0.01 1.1 ± 0.2 4.1 ± 0.1
7 3
Comparison of the total As content (Table 1) and total extracted As from
the bioaccessibility studies (Table 3), shows that nearly all the As from carrots
and beets is bioaccessible (98% and 90% respectively). However, for quinoa,
only about 40% of As is bioaccessible.
It is well known that not all the species found in the food matrix are
bioavailable (Bastias et al., 2013b). The thiol groups in certain proteinrich rice
varieties are strongly bound to As(III), which can influence its bioaccessibility
(Rahman et al., 2014b). Quinoa presents an usual protein content within the
14–22% range, that is about two or three times higher than rice. In order to
find out whether the digestion process could alter the original species present
in the vegetables (and, consequently, their toxicity risk), the salivary, gastric
and intestinal extracts of the three vegetables were analyzed by LC-ICPMS.
A comparison of the distribution of the As species present in the methanolic
extract for carrots and quinoa or in the enzymatic extract for beets (Table 2),
along with the percentage of the As species extracted after the whole
gastrointestinal process can be both found in Table 4. No significant
Table 2
 I. Pizarro et al. / Science of the Total Environment 565 (2016) 557–563 561

Methanolic and enzymatic extraction efficiency for total As in carrots, beets and quinoa, and As
species contribution (%) to total As content in both extracts. n = 3.
Vegetables Methanolic extraction efficiency Enzymatic extraction efficiency
(%) (%)
Carrots As(III): 45 ± 7 As(III): 50 ± 10
Carrots Total As: 97 ± 5 Total As: 12 ± 5 As(V): 43 ± 5 As(V): 36 ± 15

As(III): 45 ± 7 As(III) b DL Beets As(III) b DL As(III) b DL

As(V): 20 ± 5 As(V): 33 ± 7
As(V): 43 ± 5 As(V) b DL
Unknown: 70 ± 3 Unknown: 70 ± 12
Beets Total As:10 ± 2 Total As: 95 ± 9
As(III) b DL As(V): 20 ± 5 Quinoa As(III): 15 ± 4 As(III) b DL
As(V): 82 ± 9 As(V): 75 ± 7
As(V) b DL Unknown: 70 ± 3

Quinoa Total As: 99 ± 6 Total As: 7.4 ± 0.9

As(III): 15 ± 4 As(III) b DL
As(V): 82 ± 9 As(V) b DL
transformation of the original As species found in the methanolic or
enzymatic solutions seems to take place during “in vitro” gastrointestinal
digestion of the vegetables.
A previous work (Calatayud et al., 2013) showed that gastrointestinal
digestion did not alter As(V), MMA, DMA and As(III) species. When
broccoli, asparagus and spinach were soaked or boiled with As(V) and
As(III), these species were not altered after digestion and the bioaccessibility
of As species was about 100%, as in our case for carrots and beets.
The main reported studies about bioaccessibility of total and inorganic As
have been focused on rice due to its capability to preconcentrate this element,
the toxic inorganic As species present and the fact that the main staple food
of a large part of the world population is rice. However, quinoa is also a
common pseudo-cereal with high consume in South American countries and
is being spread to other countries mainly because of immigration. Frequently,
for non-severely contaminated places such as some Indian areas, As
concentration in rice is about 0.2 mg kg-1. (He et al., 2012). This is also the
average As concentration found for quinoa grown in the contaminated Chiu
Chiu area. He et al. reported that the extractable As in different types of rice
ranged from 53 to 102%, suggesting that the bioaccessibility of As might be
dependent on the individual rice samples. In our case, the average percentage
of extractable arsenic in quinoa is close to that obtained by these authors for
brown rice. However, an important difference between both staples is the fact
that the main inorganic species in rice is As(III), while in quinoa it is As(V).
3.4. Dietary As exposure estimation
Fresh carrots and beets present a water content of about 85%, while in
quinoa it is only about 10%. The average consumption of quinoa per
inhabitant in the Andean countries has been reported as 1.15–2.35 kg per year
(Vega-Galvez et al., 2010; FAO-ALADI, 2013) and around 2– 6 kg per year
for carrots and beets (Palomo et al., 2009).
Considering a maximum intake of 2.5 kg per year for quinoa, and 6 kg per
year for carrots and beets; the As content in the lyophilized
Table 3
Table 4
Comparison of the As species distribution in the methanolic extract for carrots and quinoa and
enzymatic extract for beets and the As species distribution in the whole gastrointestinal extract with
respect to the total As extracted.
Bioaccessibility of As in Chilean vegetables by in vitro gastrointestinal digestion. Results are expressed as mean ± SD (mg kg−1) in lyophilized carrots and beets and fresh quinoa. Contribution (%) of
As species to the total As content in the three gastrointestinal extracts. n = 3.
vegetables; and the mentioned water content in the fresh vegetables, dietary As
exposure estimates have been calculated according to the FAO/WHO
(FAO/WHO, 1985) approach, using the equation:
DIAs ¼ CAs CRvegetable ð1Þ
where: DIAs is the dietary intake of As (μg person−1 day−1), CAs is the As
concentration in fresh vegetables (μg kg−1) and CRvegetable is the vegetable
consumption rate (kg person−1 day−1).
The health risk of total As exposure from the tested vegetables was estimated
according to the equation:
DEAs ¼ DIAs=BWav ind ð2Þ
where: DEAs is the dietary exposure (μg kg−1 body weight day−1), DIAs is the
dietary intake of total As (μg person−1 day−1) and BWav ind is the average
individual body weight of the consumers (72 kg).
Table 5 shows the calculated DIAs and DEAs values for total As for the tested
vegetables. In the case of beets, due to the presence of organic As species, these
parameters have been calculated separately for inorganic As and for total As.
Similar DIAs and DEAs values have been obtained for the three vegetables, and
these were at the same levels as those previously reported (Sidbu et al., 2012) for
rice grain growing in As contaminated areas of India (0.016 and 0.012 μg day−1
kg−1 body weight for rural and urban population, respectively).
Furthermore DEAs values obtained for the three vegetables were much lower
than the safety standard limit of 50 μg day−1 kg−1 body weight set by Joint
FAO/WHO Expert Committee on Food Additives (JFCFA) for As
(UNEP/FAO/WHO, 1992). Therefore, considering the bioaccessibility factor for
each vegetable, the intake per person, and the concentration of total As in each
fresh vegetable (shown in Table 3), their toxicological risk is similar, although
the capability of quinoa to accumulate As seems to be higher than those of other
vegetables. However, the main As species of beets are probably arsenosugars,
considered to lack any toxicological effect in the case of seaweeds. As a result,
the influence of the toxicity of As species, in addition to their bioaccessibility,
should be considered.
Carrots Beets Quinoa

Extracted As (μg kg−1) Salivary extract 313 ± 18 289 ± 28 34 ± 4

As(III):55% Unknown:70% As(III) b
DL
As(V):30% As(V):30% As(V):85%
562 I. Pizarro et al. / Science of the Total Environment 565 (2016) 557–563

Gastric extract 135 ± 12 186 ± 21 31 ± 4

As(III):60% Unknown:60% As(III) b

DL
As(V):30% As(V):40% As(V):74%

Intestinal extract 65 ± 9 84 ± 10 13 ± 6

As(III):9% Unknown:90% As(III) b

DL
As(V):80% As(V):30% As(V):65%

Total extracted As (μg kg−1) 513 ± 45 559 ± 38 78 ± 15

Bioaccessibility (%) 98 ± 3 90 ± 4 40 ± 4

Table 5
Arsenic concentration in fresh vegetables and estimated dietary intake (DIAs) and dietary exposure (DEAs) of total As for carrots, beets and quinoa.
Vegetables [As] (μg DIAs DEAs
kg−1) (μg person−1 day−1) (μg kg−1 body weight day−1)

Carrots 78 ± 4 1.25 0.017

Beets 93 ± 6 1.50 0.021
⁎ ⁎
0.45 0.006
Quinoa 180 ± 7 1.23 0.017

⁎ Calculated for inorganic As. Appendix A. Supplementary data

Some other studies have focused on the determination of As in vegetables in
the II Chilean Region. The diversity of the areas, where different levels and
sources of contamination can be found, and the diverse As levels in water
depending on the river and the region, made it difficult to compare our results
with those reported in the Chilean bibliography (Pizarro et al., 2003b; Ferreccio
and Sancha, 2006; Romero et al., 2003). However, the main conclusions arising
from the different studies on As point out similar tendencies related to its
bioaccessibility, species present in the samples, and its environmental and health
impact.
4. Conclusions
Carrots, beets and quinoa from As-contaminated Chiu Chiu area were
analyzed to evaluate total As content and As species present. As bioaccessibility
was also investigated through in vitro gastrointestinal digestion.
Inorganic As(III) and As(V) were the only species present in carrots and
quinoa. Regarding the toxicological risk, quinoa is the vegetable able to
accumulate the highest amount of inorganic As, but its bioaccessibility is only
about 40%. On the contrary, for carrots and beets the bioaccessibility is about
100%. However, since the main As species present in beets are probably
arsenosugars, presumably less toxic, As species toxicity is a factor that should
be taken into consideration. No significant transformation of As species after in
vitro gastrointestinal digestion seems to occur.
More research is still necessary to determine the species present in the beets,
where identification is limited by both the availability of standards and the
complexity of the matrix. Furthermore, toxic arsenic species were found to be
present in the gastrointestinal extracts. This points out that the digestion process
needs to be considered for the proper assessment of the toxicological risk
associated with the ingestion of As in highly consumed vegetables.
Acknowledgements
The authors thank Universidad Complutense de Madrid, Spain (007/ 11 VIII-
2011 Cooperación al Desarrollo), the Community of Madrid, Spain (S2013/ABI-
3028), the European FEDER program and the project MECESUP U.A., Chile,
for financial support.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2016.04.199.
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